High-throughput, high-titer CHO production of miniproteins with high yields and low endotoxin levels, surpassing traditional E. coli production methods.
MiniProtein Line Platform: High-Throughput Miniprotein Production Using High-Titer CHO Gaozhan Zhang, Zhenyang Zhao, Mengjie Lu, Jiansheng Wu WuXi Biologics, NO.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_BD@wuxibiologics.com)
Miniproteins, comprised of 100-200 amino acids, are designed to mimic the structures and functions of natural counterparts. They hold significant potential in molecular biology, drug development, and biotechnology, valued for their stability, specificity, and versatility. The rise of artificial intelligence in biotechnology has sparked a growing interest in the rapid and efficient production of thousands of do novo designed miniproteins. Traditional production methods using E. coli have challenges to meet these demands, including limited throughput, complications with protein folding, and high endotoxin levels. WuXi Biologics has introduced the MiniProtein Line platform, utilizing a high-throughput (HTP), high- titer CHO expression system. This innovative platform substantially increases yields, and quality, while dramatically reducing endotoxin levels, outperforming traditional E. coli systems. Abstract Miniproteins are intricately designed to perform specific functions by folding into unique structures, making them invaluable for various therapeutic applications. Proper folding is essential for their effectiveness, as it enables the versatility required in these applications. However, traditional production methods using E. coli often encounter challenges, particularly when the proteins require disulfide bonds or specific post-translational modifications. Mammalian expression system like CHO cells provide a more efficient alternative for miniprotein production. Recognizing this, WuXi Biologics has strategically leveraged a high-titer CHO expression system in the MiniProtein Line platform, enabling HTP production of miniproteins with exceptional yields. To ensure the highest standards of purity and function, the platform integrates flexible purification techniques alongside advanced screening technologies, such as Bio-Layer Interferometry (BLI). These capabilities allow WuXi Biologics to deliver both purified miniproteins, starting from gene synthesis, and binding assay results within a timeframe of 4-5 weeks. Introduction Fig 1. HTP Miniprotein Production Workflow The MiniProtein Line platform streamlines gene synthesis, HTP production and activity assays, enabling the delivery of results within just 4-5 weeks.
Case Study 3: HTP, High-Titer Production of 76 Miniproteins in 3 mL CHO Within 2 Weeks Fig 4. SDS-PAGE analysis for two-step purification of 76 miniproteins
• 7-day transient CHO cell expression of 76 miniproteins in 24-well plates • Subsequent Ni purification in 24-well plates, followed by SEC purification
Fig 5. Average yields and purity for 76 miniproteins in each purification step
In this case study, we demonstrated the HTP production of 76 miniproteins in 3 mL of CHO cells over two weeks. Yields ranged from 0.55 mg after Ni purification to 0.16 mg after Ni-SEC purification. The final product achieved an average purity of 88%. Case Study 4: Scale-Up, High-Purity Production of a Miniprotein in 1 L CHO Fig 6. SDS-PAGE (NR/R) and SEC-HPLC analysis for scale-up production of a miniprotein
Results
Fig 2. SDS-PAGE analysis and average yields for one-step purification of 48 miniproteins Case Study 1: Super High-Titer Expression of 48 Miniproteins with 20 Different Folds
In this case study, we scaled-up the production of a miniprotein in 1 L of CHO cells. After removing fusion tags, 700 mg was collected in the Ni purification flow-through. Following SEC purification, 450 mg of the final product was obtained, with a purity of 99.4% and an endotoxin level of < 0.015 EU/mg.
The titers of most miniproteins in CHO is >100 mg/L
We extend our gratitude to all scientists from the Department of Protein Sciences at WuXi Biologics for their invaluable support during the development of Miniprotein Line platform. Additionally, we would like to thank the legal department, public relations department, and marketing department for their assistance with patent filing, poster preparation, and revisions. Acknowledgment WuXi Biologics has developed HTP, high-titer production and streamlined scale-up of miniproteins using the MiniProtein Line platform. These miniproteins are well-suited for rapid assessment of binding constants. Conclusions Method All experiments were conducted within the Protein Sciences (PS) Department at WuXi Biologics. The DNA sequence codon was optimized using WuXi Biologics’ laboratory data management system (LDMS), and the plasmid was constructed into WuXi Biologics’ proprietary vector. Subsequently, a WuXian Transient procedure was performed following SOPs, and the culture medium was collected after 7 days. The purification featured a single-step, automated method where the medium was processed through a high- throughput system and purified with magnetic beads. For column purification, the supernatant was loaded onto suitable purification columns based on titer. The purification steps included a Nickel column, his-tag cleavage, and size exclusion chromatography (SEC). The final product analysis was conducted using SDS-PAGE, SEC-HPLC, intact Mass analysis, and endotoxin detection. • The platform delivers exceptional titers ranging from 100 to 500 mg/L, ensuring HTP production and superior yields. • The final products exhibit outstanding quality, marked by high purity and low endotoxin levels. • The streamlined process significantly reduces production timelines, enabling delivery in 4-5 weeks.
This case study demonstrated HTP production of 48 miniproteins with 20 different structures through a single- step purification. With an average titer of 197 mg/L, most miniproteins reached titers over 100 mg/L in CHO cells—a remarkable achievement for molecules around 10 kDa. Case Study 2: Superior Yield and Purity in CHO Over E. coli for Miniproteins with Disulfide Bonds Fig 3. SDS-PAGE analysis and titers for 5 miniproteins expressed in CHO and E. coli
This case study evaluated the purity and titers of 5 published miniproteins with disulfide bonds expressed in CHO cells compared to E. coli. The results showed that CHO expression systems offer significant advantages, achieving much higher yields and substantially reduced aggregations.
Poster modified on 8/14/2024
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To discuss this poster: PS_BD@wuxibiologics.com
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